Technical Field
The present disclosure relates to novel, small molecule inhibitors for G protein-coupled receptor kinase 2 (“GRK2”), and methods of using the small molecules to inhibit GRK2 for the treatment of heart disease and hypertension.
Description of Related Technology
Eukaryotic cells regulate the strength and duration of signaling cascades, and therefore, must rapidly adapt to changes in their extracellular environment. G protein-coupled receptors (“GPCRs”) are the largest class of receptors in humans, and regulate nearly all aspects of eukaryotic cell physiology. GPCR activity is controlled via the phosphorylation of serine and threonine residues in GPCR cytoplasmic tails and loops by G protein-coupled receptor kinases (GRKs) (see Gurevich et al., Pharmacol Ther 133:40-69 (2012); Pitcher et al. Annu Rev Biochem 67:653-692 (1998)). The phosphorylated GPCRs recruit arrestins (see Gurevich and Gurevich Pharmacol Ther 110:465-502 (2006)), which uncouple the GPCRs from G proteins, target the receptors to clathrin-coated pits for endocytosis, and serve as adaptors for other signaling pathways (see Claing et al., Prog Neurobiol 66:61-79 (2002); Lymperopoulos Curr Pharm Des 18:192-198 (2012); Reiter et al. Annu Rev Pharmacol Toxical 52:179-197 (2012)).
GRKs can be classified in one of three subfamilies based on gene structure and homology. The vertebrate-specific GRK1 subfamily includes GRK1 (rhodopsin kinase) and 7, which are expressed in the rod and cone cells of the retina. The GRK2 subfamily, which includes GRK2 and GRK3, are Gβγ-dependent and play important roles in the heart and olfactory neurons, respectively. In particular, GRK2 phosphorylates activated β-adrenergic receptors, thereby preventing overstimulation of cAMP-dependent signaling (see, e.g., Diviani et al. J Biol Chem 271:5049-5058 (1996); Hausdorff et al., Symp Soc Exp Biol 44:225-240 (1990); Lefkowitz et al., Trends Phamracol Sci 11:190-194 (1990)). The GRK4 subfamily includes GRK4, 5, and 6. GRK5 and 6 are ubiquitously expressed. GRK5 plays an important role in heart function, albeit distinct from GRK2. All GRKs specifically recognize and phosphorylate only activated GPCRs, and phosphorylate peptide substrates derived from the activated receptors with KM values up to three orders of magnitude higher than for the full length receptor (see Palczewski et al., Biochemistry 27:2306-2313 (1988)), indicating the existence of an allosteric docking site on GRKs.
GRK2 overexpression in the heart is a biomarker for heart failure, and leads to uncoupling of heart function from sympathetic control. In the failing heart, the loss of cardiac output promotes increased levels of circulating catecholamines, resulting in severe uncoupling of βARs and a loss of inotropic reserve (see Eschenhagen Nat Med 14:485-487 (2008)). This uncoupling coincides with a 2-3 fold increase in GRK2 activity accompanied by an increase in both protein and mRNA levels (see Ungerer Circulation 87:454-463 (1993); Ungerer Circ Res 74: 206-213 (1994)). Thus, in chronic heart failure, GRKs become overexpressed and are linked to disease progression. Studies in mice overexpressing GRK2 in the heart show attenuation of isoproterenol (Iso)-stimulated contractility, reduced cAMP levels, and impaired cardiac function (see Koch et al., Science 268:1350-1353 (1995)), likely through desensitization of cardiac β1-adrenergic receptors (see Vinge et al. Mol Pharmacol 72:582-591 (2007)). Therefore, inhibition of GRK2 function could be beneficial during heart failure (see Hata et al., J Mol Cell Cardiol 37:11-21 (2004); Lymperopoulos et al., J Biol Chem 285:16378-16386 (2010)). For example, studies in animal models using the GRK2 inhibitory protein βARKct, or with cardiac-specific GRK2 gene deletion, have shown that inhibition of GRK2 or lowering expression improves heart failure outcome (see Raake et al., Eur Heart J, Jan. 19, 2012; Raake et al., Circ Res 103:413-422 (2008); Rengo et al., Circulation 119:89-98 (2009); Rockman et al., Proc Natl Acad Sci USA 95:7000-7005 (1998); Shah et al., Circulation 103:1311-1316 (2001); White et al., Proc Natl Acad Sci USA 97:5428-5433 (2000)).
Because GRK2 overexpression in the heart is a biomarker for heart failure, inhibitors of GRK2 have been developed for the treatment of cardiovascular disease. Polyanionic GRK2 inhibitors, such as heparin and dextran sulfate, however, are nonselective (see Benovic et al., J Biol Chem 264:6707-6710 (1989)). Although the natural product, balanol (FIG. 1A), was found to inhibit GRK2 in the low nanomolar range, is a non-selective inhibitor of the protein kinase A, G and C family (AGC kinases) (see Setyawan et al., Mol Pharmacol 56:370-376 (1999); Tesmer et al., J Med Chem 53:1867-1870 (2010)). Other inhibitors of GRKs have also been described, but these either have poor potency (see lino et al., J Med Chem 45:2150-2159 (2002), low selectivity (see Winstel et al., Biochem Pharmacol 70:1001-1008 (2005)), or non-drug like properties (see Benovic et al., Science 246:235-240 (1989)). For example, a class of heterocyclic compounds, such as Takeda103 (FIG. 1B), which is selective for the GRK2/3 subfamily (see PCT publication No. WO 2007/034846, incorporated herein by reference in its entirety) have been shown to bind in the active site of the enzyme (see Thal et al., Mol Pharmacol 80:294-303 (2011)). Paroxetine also was found to selectively inhibit GRK2 (FIG. 1C), mediating beneficial responses in animal models, but only exhibits micromolar potency (e.g., −20 μM) (see Thal et al., ACS Chem Biol 7:1830-1830 (2012)). A family of ROCK inhibitors (e.g., GSK180736A (FIG. 1D)) also act as GRK2 inhibitors (see Goodman et al., J Med Chem 50:6-9 (2007); Sehon et al., J Med Chem 51:6631-6634 (2008)).
Therefore, there is a need for GRK2 inhibitors that exhibit high potency, high selectivity, and good pharmacokinetic properties for the treatment and prevention of cardiac disease.